Cognitive decline in post-COVID-19 syndrome does not correspond with persisting neuronal or astrocytic damage

Cognitive impairment is the most frequent symptom reported in post-COVID-19 syndrome (PCS). Aetiology of cognitive impairment in PCS is still to be determined. Neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) are increased in acute COVID-19. Their role as biomarkers in other neurological disorders is under debate. We analysed serum levels of NfL and GFAP as markers for neuronal and astrocytic damage in 53 patients presenting to a PCS Neurology outpatient clinic. Only individuals with self-reported cognitive complaints were included. In these individuals, cognitive complaints were further assessed by comprehensive neuropsychological assessment (NPA). Patients were categorized into subgroups of subjective cognitive decline, single domain impairment, or multi-domain impairment. Serum NfL was in normal range, however an increase of serum GFAP was detected in 4% of patients. Serum NfL and GFAP levels correlated with each other, even when adjusting for patient age (r = 0.347, p = 0.012). NPA showed deficits in 70%; 40% showing impairment in several tested domains. No significant differences were found between serum NfL- and GFAP-levels comparing patients with subjective cognitive decline, single domain impairment, or multi-domain impairment. Persistent neuronal or astrocytic damage did not correlate with cognitive impairment in PCS.

Symptoms in post-COVID-19 syndrome (PCS), also referred to as post-acute sequelae of COVID-19, are manifold and include neurological and neuropsychiatric symptoms 1 .One of the most frequently reported symptoms in patients with PCS are cognitive complaints, which can be evaluated by neuropsychological assessment (NPA) [2][3][4][5] .To date, aetiology of neurological symptoms in PCS is still unknown.
Furthermore, elevation of those markers in hospitalized COVID-19 patients have been associated with longterm neurological symptoms including cognitive impairment 16 .In contrast, in a longitudinal analysis of blood GFAP-and NfL-levels of COVID-19 patients, normal levels were noted 6 months after acute SARS-CoV-2 infection following an initial peak, although half of the patients reported persistent neurological symptoms 17 .of the medial cerebral artery (n = 1); occipital microbleeds (n = 1); unspecific white matter lesions (n = 4); slightly prominent parietal sulci (n = 1) and leukoaraiosis stable compared to scans from before COVID-19 (n = 1).NPA confirmed cognitive deficits in 37 patients (70%).16 patients (30%) had deficits in only one domain, while 21 (40%) had deficits in two or more domains.Deficits were detected most frequently in divided attention (n = 18, 34%), followed by selective attention (n = 14, 26%) and visual retrieval (n = 14, 26%).A predominant phenotype of cognitive deficits was not detectable, as shown in an Upset Plot of deficits in NPA and their intersections (Fig. 1).MoCA total scores correlated with the NPA category (r = − 0.325, p = 0.017).
Median value of serum NfL was 7.4 pg/ml (interquartile range 6.0-10.0pg/ml), and 89.7 pg/ml for GFAP (interquartile range 67.0-107.6 pg/ml).Considering established age-specific reference levels for serum NfL and GFAP (Supplemental Tables S1 and S2) 20,21 , all detected NfL levels were within the upper limit of normal and elevated serum GFAP levels were found in only two individuals (4%).

Discussion
Elevation of GFAP and NfL has been reported in the acute phase of COVID-19, particularly in severe cases 10,14,15 .A Chinese cohort study showed that severe COVID-19 is associated with long term cognitive impairment 12 months after acute disease 22 .In a cohort of hospitalized COVID-19 patients from the UK, significantly higher values of NfL and GFAP were measured compared to controls in the acute phase 23 .Patients who developed neurological complications such as cerebrovascular incidents still presented elevated NfL values in the late convalescent phase, which was defined as more than 6 weeks after first positive SARS-CoV-2 PCR 23 .Cognitive www.nature.com/scientificreports/impairment after severe COVID-19 might be attributed to neuronal damage in the acute phase.Whether these deficits and their aetiology differ from cognitive impairment in post-intensive care syndrome (PICS) 24 is still to be determined and needs to be the subject of further studies.Our data does not support the hypothesis of persisting neuronal or astrocytic damage as the leading cause of cognitive decline in PCS.This is in line with previous reports showing normalization of GFAP and NfL levels 6 months after acute COVID-19 17 and normal blood NfL-and GFAP levels in patients with persisting headache in PCS 25 .As such, serum NfL and GFAP appear not to be sensitive biomarkers for cognitive impairment in PCS.Interestingly, recent findings in a Spanish cohort of PCS patients with subjective cognitive complaints revealed reduced hippocampal grey matter volume and elevated NfL and GFAP levels compared to controls but did not apply age-adjusted reference values 26 .Implication for clinical assessment as biomarker usage is questionable to date.
In our cohort, most patients had a mild COVID-19 course.Here, other pathomechanisms than those discussed after severe COVID-19 are of potential relevance and under debate 27 .Previous findings suggest that a persistent central nervous system infection with SARS-CoV-2 may not be the cause of neurological or neuropsychiatric symptoms in PCS 28 .Patients with cognitive impairment in PCS showed higher CCL11/eotaxin-1 plasma values than patients with non-cognitive PCS symptoms, a cytokine that leads to specific reactivity of hippocampal microglia and impaired hippocampal neurogenesis in mice after systemic application 29 .Neuroinflammation and microglial reactivity and their role in cognitive impairment in PCS should be further evaluated.Furthermore, the role of autoimmunity in cognitive decline in PCS is of concern, since brain-binding autoantibodies can be found in CSF of patients with PCS and are associated with cognitive impairment 30 .
Although-as predefined per study inclusion criteria-all patients in our study reported subjective cognitive complaints, less than a third had abnormal MoCA values.Via comprehensive NPA, deficits were detectable in almost 70%, with approximately 40% having deficits in several neuropsychological domains.Interestingly, 30% did not show any deficits including NPA.This could be due to higher premorbid cognitive capacities and therefore higher premorbid test scores of those patients, with their current test scores still ranging within normalised means.Since neuropsychological data of our cohort prior to COVID-19 is lacking, this remains hypothetical.The discrepancy between subjective cognitive complaints and objective cognitive impairment in NPA has been previously reported for PCS 31 .A PCS specific phenotype restricted to specific neuropsychological deficits could not be determined by our study.
Frequent concomitant symptoms were fatigue and depressive symptoms since COVID-19.ME/CFS is commonly associated with cognitive impairment and also presenting with a heterogeneous profile in NPA 32 .Cognitive dysfunction is also a leading symptom in depression.In depressive patients, NPA shows deficits in several domains, even after remission 33 .Whether cognitive impairment in PCS is a singular entity or the result of other, potentially multifactorial, determinants, needs to be the subject of further studies.
The strengths of our study are the combination of biomarker analyses with a comprehensive neuropsychological assessment as well as the inclusion of patients at two centres.To minimize bias of our data, inclusion and exclusion criteria were applied to analyse alterations in NfL, GFAP levels and results in NPA most likely caused by post-COVID-19 syndrome and not by other disorders.Limitations of our study are the lack of sequential biomarker and neuropsychological assessments.While there is data showing improvement of cognitive deficits over time, other cohorts found a persisting decrease of processing speed over a 6-month period 34,35 .To provide further longitudinal neuropsychological testing in PCS, some participants of our cohort from the study centre in Cologne were evaluated in neuropsychological follow ups.Due to ongoing analyses, results of these follow ups will be published separately.Another limitation is the assessment of only two biomarkers of possible interest.NfL and GFAP were selected in this study due to the reported elevation in acute COVID-19.Not evaluated in this study were several other cytokines, chemokines and hormones such as tumor necrosis factor, CXCL9 and cortisol, which have been reported to be altered in PCS and could potentially serve as biomarkers for PCS [36][37][38] .

Study population
Patients (n = 198) presenting to the PCS Neurology outpatient clinics in Berlin and Cologne were screened by a physician specialized in Neurology or Psychiatry for study eligibility.If available, cranial MRI scans, which were performed due to cognitive complaints after COVID-19, were evaluated for causative structural cerebral changes.Only patients fulfilling the PCS criteria by the WHO Delphi consensus 39 and with a confirmed diagnosis of SARS-CoV-2 infection (either positive PCR testing for SARS-CoV-2-RNA or positive testing for SARS-CoV-2 antibodies prior to vaccination), and who predominantly reported of cognitive complaints following COVID-19 were included in this study.Patients with pre-existing neurological or psychiatric diagnosis, especially prior affective disorders, were excluded, resulting in 53 patients participating.All participants gave informed written consent.This study was approved by the ethics committee of Charité-Universitätsmedizin Berlin (EA2/066/20) and the ethics committee of the University of Cologne (20-1501).The study was conducted in accordance with the declaration of Helsinki.

Neuropsychological assessment
The Montreal Cognitive Assessment Scale (MoCA) was performed to screen for cognitive impairment 40 .Further NPA comprised well-validated tests covering the neuropsychological domains of attention, executive functioning, verbal memory, language and visual retrieval (Table 2).Selective attention was evaluated via the Trail Making Test (TMT) A or the Test of Attentional Performance (TAP) tonic alertness 41,42

GFAP and NfL analysis
Serum NfL and GFAP levels were measured in duplicates using the single molecule array HD-X analyzer (Quanterix, Boston, MA) and the NF-light Advantage Kit and GFAP Discovery Kit according to the manufacturer's protocol.

Data analysis
Statistics were computed using SPSS (IBM Corp. 2013 SPSS Statistics for Windows, version 29.0).Spearman's rho was performed for bivariate correlation testing.Partial rank correlation was used for non-parametric partial correlation testing.Welch's t-Test was used to analyse differences in NfL or GFAP levels in patients with normal and abnormal MoCA scores.One way ANOVA was used to analyse differences in NfL or GFAP levels in patient subgroups.An α-level of 0.05 was chosen for determination of statistical significance.Upset Plots were created with Python 50 .

Figure 1 .
Figure 1.Deficits in neuropsychological assessment and their intersections plotted as an Upset Plot.

Figure 3 .
Figure 3. Boxplots of levels of neurofilament light chain (NfL) or glial fibrillary acidic protein (GFAP) in different cognitive subgroups.(a) Levels of NfL in the groups of normal and abnormal score in the Montreal Cognitive Assessment (MoCA).(b) Levels of GFAP in the groups of normal and abnormal score in the MoCA.(c) Levels of NfL in the groups of subjective cognitive decline (SCD), single domain impairment (SDI) and multi-domain impairment (MDI) according to neuropsychological assessment (NPA).(d) Levels of GFAP in the groups of SCD, SDI and MDI according to NPA.
45,46,48,49]om/scientificreports/evaluatedvia TMT B or omissions in the TAP divided alertness testing41,42.Working memory components of executive function was analysed by testing of the digit span backwards (DSB) of the Wechsler Memory Scale-Revised Manual (WMS-R)43.For verbal memory, the German version of the Auditory Verbal Learning Test (Verbaler Lern-und Merkfähigkeitstest; VLMT) or the word list learning of the Consortium to Establish a Registry for Alzheimer's Disease neuropsychological test battery (CERAD) was assessed[44][45][46]. Memory span (VLMT-first trial [D1]; CERAD-Word List Learning-first trial), immediate recall (VLMT-Trial 1 to 5 [∑D1-5]; CERAD-Word List Learning Total) and delayed recall (VLMT-Trial 7 [D7]; CERAD-Word List Recall) were evaluated[44][45][46].Language was assessed via evaluation of semantic verbal fluency as part of the Regensburger Wortflüssigkeitstest (RWT), a German version of the Controlled Oral Word Association test or the CERAD battery[45][46][47].Visual retrieval was evaluated either by the Rey-Osterrieth Complex-Figure-Test (ROCF)-delayed recall, by the Wechsler Memory Scale (WMS) IV subtest visual memory II or by the CERAD constructional praxis-delayed recall45,46,48,49.Test results were interpreted as impaired, if the achieved results were less than one standard deviation below test-specific age-, educational level-, and sex-adjusted normalised means.Results of NPA were categorized as subjective cognitive decline (SCD), if NPA showed no impaired results, since all patients self-reported cognitive complaints; single-domain impairment (SDI), if only one of the analysed domains was impaired; or multi-domain impairment (MDI), if two or more domains were concerned.